Design and Synthesis of Novel HIV-1 NNRTIs with Bicyclic Cores and with Improved Physicochemical Properties

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent cornerstones of current regimens for treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, NNRTIs usually suffer from low aqueous solubility and the emergence of resistant viral strains. In the present work, novel bicyclic NNRTIs derived from etravirine (ETV) and rilpivirine (RPV), bearing modified purine, tetrahydropteridine, and pyrimidodiazepine cores, were designed and prepared. Compounds 2, 4, and 6 carrying the acrylonitrile moiety displayed single-digit nanomolar activities against the wild-type (WT) virus (EC50 = 2.5, 2.7, and 3.0 nM, respectively), where the low nanomolar activity was retained against HXB2 (EC50 = 2.2–2.8 nM) and the K103N and Y181C mutated strains (fold change, 1.2–6.7×). Most importantly, compound 2 exhibited significantly improved phosphate-buffered saline solubility (10.4 μM) compared to ETV and RPV (≪1 μM). Additionally, the binding modes of compounds 2, 4, and 6 to the reverse transcriptase were studied by X-ray crystallography.


■ INTRODUCTION
Reverse transcriptase (RT) of human immunodeficiency virus (HIV) is essential for viral replication due to its responsibility for the conversion of viral RNA genome into DNA. 1 Thus, RT represents an important and established target among anti-HIV-1 therapies to treat acquired immune deficiency syndrome. 2−5 RT contains two distinct druggable sites: the polymerase catalytic site that is available for nucleoside reverse transcriptase inhibitors (NRTIs) and their acyclic nucleoside phosphonate analogues (ANPs), and the allosteric binding site available for non-NRTIs (NNRTIs). 6,7 The aforementioned drug classes play a crucial role in modern highly active antiretroviral therapy, a regimen based on a combination of at least two classes of antiretroviral drugs. 8 The main advantages of NNRTIs are their lack of requirement for metabolic activation, low nanomolar potency, and noncompetitive kinetics of binding to RT. Clinical use of first-generation NNRTIs, nevirapine and delavirdine, was limited by their low genetic barrier to resistance and toxicity. 2,9,10 Efavirenz is a component of the first once daily single-tablet regimen to treat HIV-1; however, efavirenz-based regimens have a high rate of central nervous system side effects, which affect the overall tolerability. 11 These issues with first-generation NNRTIs were partly circumvented in secondgeneration inhibitors, including the diarylpyrimidine (DAPY) structural family, namely etravirine (ETV) and rilpivirine (RPV), and lately the 2-pyridinone derivative doravirine (DOR). 12,13 NNRTIs target an allosteric binding pocket which is situated about 10 Å from the polymerase catalytic site. Although NNRTIs include a wide range of structurally diverse scaffolds, their interactions with RT show similar binding modes. A "butterfly-like" model with a hydrophilic center ("body") and two hydrophobic moieties ("wings" or "arms") is a typical conformation of the first-generation NNRTIs. Alternatively, the RT-bound conformation of the DAPY family resembles a "horseshoe" or "U" shape. The inherent flexibility of ETV and RPV has been hypothesized to allow them to adapt to the binding pocket and provide the conformational adaptability to evade resistance against a wide range of mutations. 14,15 Common NNRTI resistance mutations include Y181C and K103N. These mutations continue to show a high amount of transmitted drug resistance. 16 The DAPY class typically suffers from poor aqueous solubility, which can contribute to low bioavailability and drug-like properties. Hence, the development of novel DAPYlike inhibitors with high potency against the WT virus and resistant viral strains, as well as improved aqueous solubility, is highly desirable. 17−24 Numerous publications 17,25−37 and reviews 12,38,39 on DAPY NNRTIs have been recently published. In several cases, another (hetero)cycle was attached to the pyrimidine central core of molecules to form fused bicyclic NNRTIs in order to explore the entrance channel of the NNRTI binding pocket. 25,28,35,37 In our current work, we decided to substitute the pyrimidine core of ETV and RPV ( Figure 1) with modified bicyclic cores in order to introduce an additional polar moiety, which should result in increased aqueous solubility. Furthermore, the second ring (ring B) of the bicyclic cores may also prove advantageous from the conformational viewpoint: it is expected to lock the inhibitors in the desirable U-shape to introduce potency (especially for the six-membered analogues), 40 while not being aromatic, it still keeps certain flexibility to accommodate changes in the binding pocket (to preserve the resistance profile). Since the size of ring B can, to some extent, influence the mutual position of the two lateral hydrophobic arms, analogues containing five-membered (compounds 1 and 2), sixmembered (3 and 4), and seven-membered (5 and 6) rings were designed (Figure 1).

■ RESULTS AND DISCUSSION
Chemistry. We started with the synthesis of bicyclic NNRTIs containing the six-membered ring, that is compounds 3 and 4 (Figure 1), since such compounds were expected to have the appropriate "horseshoe" conformation and, thus, to be the most potent inhibitors of the whole series.
First, compound 3 bearing the cyano group on arm I, similarly to ETV, was prepared in four steps from commercially available 4-amino-3,5-dimethylbenzonitrile (7, Scheme 1). Treatment of 7 with pyrimidine derivative 8 and DIPEA in dioxane afforded diarylamine 9 (55% yield), which was then alkylated with ethyl bromoacetate in DMF to give glycinate 10 in a 21% yield. Subsequent introduction of arm II using 4aminobenzonitrile under microwave conditions gave intermediate 11 (54%), which was reductively cyclized with Fe/ HCl to afford the target compound 3 in a 27% yield.
We decided to prepare the six-membered ring RPV analogue, that is compound 4 (Figure 1), in a similar manner to compound 3. Thus, treatment of 4-bromo-2,6-dimethylaniline (12) with pyrimidine 8 and DIPEA in dioxane, followed by alkylation with ethyl bromoacetate in DMF, afforded   compound 13 in a 57% yield over two steps (Scheme 2). Subsequent introduction of arm II using 4-aminobenzonitrile and reductive cyclization with either Fe/HCl or H 2 /Ra−Ni gave the bicyclic intermediate 14 in 8 and 14% yields (two steps), respectively. Our initial approach to the bicyclic RPV analogue, that is compound 4, relied on an introduction of the sensitive cyanovinyl moiety via Mizoroki−Heck coupling between acrylonitrile and aryl bromide 14. However, the cyanovinylation of 14 by Mizoroki−Heck coupling proved to be inefficient. For example, using the reaction conditions previously used for the preparation of RPV 41 afforded a complex reaction mixture with less than 10% of the desired product 4 [determined by ultra-performance liquid chromatography−mass spectrometry (UPLC−MS)]. Ensuing screening of some 20 alternative reaction conditions for Mizoroki− Heck reaction (various catalysts and catalyst loadings, various reaction temperatures) led to a slight improvement only: when bulky Pd[P(t-Bu 3 )] 2 was used stoichiometrically in DMF with TEA as a base, desired product 4 was obtained in a 35% yield with 80% purity. Furthermore, isolated product 4 was an inseparable mixture of E-and Z-isomers (2/3) in favor of the undesired Z-isomer. An attempted isomerization of the cyanovinyl double bond (using I 2 , hv) was not successful. It became apparent that a new synthetic route for target compound 4, as well as other bicyclic RPV analogues, was required.
Preliminary biological data obtained for target compound 3 and for bromo intermediate 14 revealed low nanomolar activity against the WT virus, high potency against Y181C and K103N mutated strains, and also significantly improved aqueous solubility. 42 Encouraged by these promising data, we decided to finish the easier-to-make ETV series first.
The synthesis of five-membered ETV analogue 1 ( Figure 1) started with the treatment of intermediate 9 with 4aminobenzonitrile in i-PrOH to give compound 15 in a 62% yield (Scheme 3). The nitro group of 15 was then reduced by tin(II) chloride (to give amine 16 in an 82% yield), followed by the closure of the five-membered ring by treatment with carbonyldiimidazole (CDI) 43 in DCM to give the desired compound 1 (Scheme 3).
In order to synthesize the seven-membered ETV analogue 5 (Figure 1), starting aniline 12 was alkylated with ethyl 3bromopropionate (to give 17 in a 46% yield), followed by nucleophilic aromatic substitution with 2,4-dichloro-5-nitropyrimidine to afford compound 18 in a 17% yield (Scheme 3). The reaction of 18 with 4-aminobenzonitrile in i-PrOH gave the intermediate 19 in a 55% yield. Aryl bromide 19 was then converted into the corresponding nitrile by palladiumcatalyzed cyanation, and subsequent reductive cyclization with tin(II) chloride and Sc(OTf) 3 gave the desired pyrimidodiazepin-6-one analogue of ETV, compound 5 (Scheme 3), in a 54% yield over two steps. Scheme 3. Synthetic Route for ETV Analogues 1 and 5 a Our next goal was to develop a successful synthetic route toward the bicyclic RPV analogues 2, 4, and 6 ( Figure 1). The installation of the cyanovinyl moiety using Mizoroki−Heck cyanovinylation on compound 14 (Scheme 2) in the last step of the first-generation synthesis failed. Thus, in the secondgeneration synthesis, the Horner-Wadsworth-Emmons reaction using (cyanomethyl)phosphonate and corresponding aldehyde intermediates I (Figure 2) was chosen for the introduction of the sensitive cyanovinyl moiety in the late stage of the novel synthetic approach. The desired aldehydes I and benzyl alcohols II can be prepared from compound 20 ( Figure  2), which was identified as a suitable precursor for the preparation of the target bicyclic RPV analogues.
Synthesis of precursor 20 (Scheme 4) started with the treatment of 4-bromo-2,6-dimethylaniline (12) with the Vilsmeier-Haack-Arnold reagent to give dimethylformimidamide 21 44,45 in a quantitative yield. Compound 21 was then formylated using n-butyllithium/DMF (to yield the aldehyde 22 44 ) and subsequent reduction of the aldehyde group with NaBH 4 in MeOH gave the corresponding alcohol 23 in an 81% yield over two steps. The removal of the protecting group with LiOH/2-aminoethanol afforded amino alcohol 24 (75%), 45 which was then readily silylated under standard conditions to provide the desired aniline 20.
Aniline 20 (Scheme 4) was then treated with 2,4-dichloro-5nitropyrimidine (8) and DIPEA in dioxane to give secondary amine 25 (88%), 43,46 which was then alkylated by ethyl bromoacetate to yield glycinate 26 in a 66% yield. Nucleophilic aromatic substitution with 4-aminobenzonitrile was performed on compound 26 under MW-assisted heating to introduce arm II, followed by reductive cyclization with tin(II) chloride as a reducing agent and with a catalytic amount of scandium(III) trifluoromethanesulfonate to afford tetrahydropteridine derivatives 27 and 28 in 30 and 13% yields (over two steps), respectively. The triisopropylsilyl (TIPS) protecting group was then removed with TBAF in order to convert derivative 27 to 28 in an 82% yield.
Intermediate 25 was heated with 4-aminobenzonitrile under MW-assisted conditions to give compound 29 in a 57% yield (Scheme 5). The nitro group of 29 was reduced with tin(II) chloride to yield amine 30 (89%), which then underwent a cyclization with CDI 43 to give five-membered intermediate 31 in a 91% yield. Finally, the TIPS protecting group was removed from derivative 31 using TBAF to give benzyl alcohol 32 in an 87% yield.  All attempts to alkylate intermediates 20 and 25 with ethyl 3-bromopropionate, methyl acrylate, 3-oxopropanoate, or 3,3diethoxypropanoate failed, and also an attempted hydrolysis� Arndt-Eistert homologation sequence on compound 26 was futile. Thus, we decided to introduce the propionate moiety (en route to target seven-membered analogue 6, Figure 1) earlier in the synthesis.
Hence, the above-prepared intermediate 17 (Scheme 3) was selected as a suitable starting material. Since an attempted direct introduction of the formyl moiety by lithiation/trapping with DMF failed due to the retro-aza-Michael addition, compound 17 was subjected to a palladium-catalyzed cyanation, giving aryl nitrile 33 in an 87% yield (Scheme 6). The nitrile 33 was then reduced by aqueous formic acid over Ra−Ni to yield aldehyde 34 (75%), which was further reduced with NaBH 4 to the corresponding alcohol 35 in an 85% yield. Surprisingly, silylation of alcohol 35 under common reaction conditions (e.g., TIPSCl and imidazole in DMF or TIPSOTf and 2,6-lutidine in DCM) failed. Fortunately, alcohol 35 afforded silyl ether 36 on treatment with TIPSCl and silver nitrate in pyridine in an excellent yield. 47 Silyl ether 36 then reacted with 2,4-dichloro-5-nitropyrimidine (8) to yield β-alaninate 37 (Scheme 6). Microwave-assisted nucleophilic aromatic substitution introduced the 4-aminobenzonitrile arm, and subsequent reductive cyclization gave pyrimidodiazepines 38 and 39 in 17 and 20% yields, respectively. Finally, the triisopropylsilyl protecting group of 38 was removed by TBAF to give the desired alcohol 39 in an 86% yield.
With the advanced intermediates 28, 32, and 39 in hand, oxidation of the benzyl alcohols to the corresponding aldehydes, followed by the Horner-Wardsworth-Emmons reaction, remained to be performed in order to obtain RPV analogues 2, 4, and 6 ( Figure 1), respectively. The oxidation was optimized using compound 28. The use of activated MnO 2 in DMF at 25°C led to the oxidation of benzyl alcohol to the corresponding aldehyde but also resulted in a hydroxylation of α-position of the lactam moiety (determined by 2D NMR and UPLC−MS). This finding is in accordance with the literature, 48 where a similar system was oxidized with MnO 2 in a DMSO/H 2 O mixture at an elevated temperature, yielding the corresponding bis-lactam. An attempted oxidation of 28 with Dess-Martin periodinane in NaHCO 3 -buffered DCM at 0°C gave an inseparable mixture of the desired product 41 (Scheme 7) and the same hydroxylated by-product as during the previous oxidation with MnO 2 (determined by UPLC− MS). Oxidation of 28 with pyridinium chlorochromate in DCM at 0°C did not lead to the desired compound 41, and the main product was identified as the corresponding carboxylic acid (UPLC−MS). However, employing Parikh− Doering conditions led to a clean formation of desired aldehyde 41 in a 68% yield (Scheme 7). Similarly, aldehydes 40 and 42 were prepared in high yields (73 and 74%, respectively) from benzyl alcohols 32 and 39, respectively. Finally, the Horner−Wardsworth−Emmons reaction of aldehyde 41 with deprotonated diisopropyl (cyanomethyl)phosphonate in DME afforded the target tetrahydropteridine analogue 4 as a 50/1 mixture of E/Z isomers in a 64% yield.  Analogously, target compounds 2 (E/Z = 10/1, 77%) and 6 (E/Z = 19/1, 72%) were prepared from the aldehyde intermediates 40 and 42, respectively.
The numbering system used for description of NMR spectra of the prepared compounds is depicted in Figure 3.

Biology.
Anti-HIV-1 Biological Evaluation. The prepared compounds were tested for their anti-HIV-1 activity (Table 1). A viral cytopathic effect assay in MT-4 cells with HIV-1 IIIB virus was used to determine compound potency (EC 50 , 50% cell viability) in protecting virus induced cell killing. Compound cytotoxicity was assessed in MT-4 cells in the absence of virus (CC 50 , 50% cytotoxicity). The synthetic intermediates with modified arm I, that is, compounds 28, 32, 39, and 40−42, did not exhibit appreciable potency against the WT virus and, thus, were not profiled further. The designed compounds bearing the cyano group (ETV analogues 1, 3, and 5) and cyanovinyl moiety (RPV analogues 2, 4, and 6) at arm I were further profiled with HXB2 wild-type virus, K103N, and Y181C mutations to assess Fold-Change (FC) in potency against these resistance mutations. The bicyclic derivatives 2, 4, and 6 with the cyanovinyl arm I were equipotent (halfmaximal effective concentration, EC 50 = 2.2−2.8 nM) against WT/HXB2 and, in general, were more potent than their cyano arm I comparators 1, 3, and 5 (Table 1). ETV contains bromo and amino substitutions on the DAPY core. These substitutions have previously been shown to alter the position of ETV compared to RPV in the pocket. 15 Likely, with the smaller cyano arm I in 1, 3, and 5, the cores could be further elaborated to optimize the compound position in the pocket and to improve potency. The six-membered bicyclic analogue 3 was the most potent from the cyano-arm I series (ETV analogues), with EC 50 values of 8.1−8.6 nM against both wildtype viruses. Interestingly, six-membered bicyclic RPV analogue 4 retained the best potency against K103N (FC 1.2x) and Y181C (FC 2.0x) mutations compared to the other compounds tested.
Drug-like Properties. By creating the B ring in the core of DAPY analogues and introducing a polar moiety, improvements in solubility at pH 7 were observed compared to ETV and RPV. The compounds bearing five-membered ring had modestly higher solubility than those with six-and sevenmembered rings. Surprisingly, compound 4 had markedly reduced solubility at pH 2 compared to the other compounds.
Compounds were further tested in a Madin−Darby Canine Kidney (MDCK) assay to evaluate their potential for oral absorption. ETV and RPV have moderate permeability (P app ) in the MDCK assay, with RPV being slightly better than ETV. The improved pH 7 solubility of compounds 1−6 did not have a substantial effect on P app . Additionally, there was no trend for better permeability when comparing arm I versus arm II when matched with the same core. The size of the B ring had the greatest influence on P app with the trend being five-membered > six-membered > seven-membered rings (Table 1).
Next, microsome stability was profiled in purified human liver microsomes (HLM). In general, the predicted intrinsic clearance (CL int, pred ) was moderate and comparable to RPV (Table 1).
X-ray Crystallography Studies. HIV-1 RT was co-crystallized with cyanovinyl compounds 2, 4, and 6 to determine the effect of the size of ring B on binding in the NNRTI pocket ( Figure 4 and Table S1). In general, changing the core size produced the same binding mode between compounds, that is the positioning of the core is comparable, which locates arms I and II in the same place in the pocket ( Figure 4). All three compounds maintain a hydrogen bond between the aniline nitrogen and the K101 backbone carbonyl ( Figure    The potential for a second hydrogen bond between the pyrimidine core nitrogen and the K101 backbone nitrogen varies between structures. This distance is longest with the fivemembered ring at 3.4 Å, suggesting that it likely does not contribute significantly to binding affinity. The six-and sevenmembered structures show this distance to be 3.2 Å, which is somewhat long for a hydrogen bond but implies some contribution to binding affinity. Due to an opening and solvent-accessible area around the core, there is water observed interacting with ring B in the five-and six-membered structures of compounds 2 and 4, but not with the seven-membered ring B of compound 6. This water is further coordinated by E138 from the p51 subunit. Previously, E138 was shown to form a salt bridge with K101 when RPV was bound in the pocket ( Figure 4D). In these structures, this salt bridge is disrupted for the five-and six-membered ring B but is present for the seven-membered ring B ( Figure 4A−C). Thus, it appears that E138 can either form a water-mediated bond to the NNRTI core or a salt bridge to K101 but not both. E138K is a known resistance mutation to RPV and ETR that is often observed with M184I; however, it confers only 2−3 fold resistance in cell culture. 49 The implications of whether E138 forms a bond with a water or cross-subunit salt bridge are unclear. The size of ring B changes the distance between arms I and II. As measured from the terminal nitrogens in arms I and II, the five-membered ring B produces a distance of 7.5 Å ( Figure  4A) compared to RPV (PDB code: 3MEE) at 6.3 Å ( Figure  4D). Increasing the ring size subsequently leads to shorter distances, closer to RPV, where the six-and seven-membered rings are 7.3 and 6.8 Å apart, respectively ( Figure 4B,C).
The co-crystal structure with compound 2 shows the most dramatic change in protein conformation. Notably, the side chain of Y188 rotates away from Y181 and sits more parallel to the cyanovinyl ( Figure 4D,E). To allow this shift, sheets β9 and β10 move, resulting in the Cα atom of D185 moving 4.7 Å compared to the RPV bound structure ( Figure 4D). Because of this, several amino acid residues become disordered at the periphery of the pocket, markedly from residues 217 to 222. These protein movements are possibly due to the position of the carbonyl in ring B and/or the wider distance between the arms in the horseshoe shape. To accommodate the carbonyl, the Cβ atom in Y181 moves 1.2 Å compared to the position with RPV bound. The measured distance from the carbonyl oxygen to the Cβ is 3.5 Å. Taken together, this illustrates the flexibility that is possible with wild-type RT.

■ CONCLUSIONS
A series of novel DAPY-based NNRTIs were designed based on an addition of a second non-aromatic ring to the pyrimidine core. These bicyclic NNRTIs contained either a fivemembered (purine core), six-membered (tetrahydropteridine core), or seven-membered ring (pyrimidodiazepine core), and either a cyano group (etravirine analogues) or a cyanovinyl moiety (rilpivirine analogues) on arm I (i.e., the o,odimethylphenyl moiety). The synthesis of target compounds via key aldehyde intermediates I was developed and optimized, using the Horner-Wardsworth-Emmons reaction in the last synthetic step. The aldehyde functionality in intermediates I offers a convenient handle for future diversification of arm I and, thus, an entry into a broader SAR study of the bicyclic NNRTIs.
All six target compounds proved to be potent antiviral agents targeting HIV-1 reverse transcriptase. Especially the cyanovinyl derivatives, that is RPV analogues 2, 4, and 6, displayed singledigit nanomolar activities against the wild-type virus (EC 50 = 2.6−3.0 nM) and low nanomolar activity against the K103N and Y181C mutated strains (Fold-Change 1.2−6.7x). All final compounds also exhibited improved phosphate-buffered saline solubility (1.4−10.4 μM) compared to ETV and RPV (≪1 μM), where the five-membered RPV analogue had the highest solubility of 10.4 μM. Furthermore, HIV-1 RT was cocrystallized with all cyanovinyl compounds, that is, derivatives 2, 4, and 6. The structures reveal that as the size of ring B changes, the distance between arms I and II changes too. Additionally, since ring B is non-aromatic and somewhat flexible, all three cyanovinyl derivatives revealed analogous binging to the RT pocket and similar anti-HIV-1 potency (EC 50 = 2.6−3.0 nM). The attachment of the second ring to the pyrimidine core of DAPY-based NNRTIs demonstrates successful structural modifications with improved drug-like properties of the inhibitors, and the prepared compounds represent an important starting point for the future design of novel anti-HIV-1 agents. ■ EXPERIMENTAL SECTION Chemistry. General Information. Unless otherwise stated, all reactions were performed under an argon atmosphere utilizing standard Schlenk techniques. Glassware was dried by heating with a heat gun under high vacuum. Solvents were evaporated at 40°C/2 mbar. Dry tetrahydrofuran was distilled from lithium aluminum hydride pellets. Acetonitrile, dimethylformamide, and dioxane were degassed by bubbling with Ar for 30 min and dried by passing through activated alumina columns in the Pure Solv system (Innovative Technology, Inc.). Ethanol and isopropanol were distilled from 3 Å molecular sieves. Other dry solvents were purchased from commercial suppliers (Sigma-Aldrich, Acros Organics). Reagents were purchased from the following commercial vendors: Sigma-Aldrich, Merck, Acros Organics, Fluorochem, Alfa Aesar, and Fluka. Microwave experiments were performed in 10 or 30 mL vials with a CEM discover (Explorer) apparatus operating at the frequency of 2.45 GHz with continuous irradiation power from 0 to 300 W. Reaction progress was routinely monitored by analytical TLC on silica gel-precoated aluminum plates with fluorescent indicator (Merck 60 F 254 ) and/or UPLC−MS on a Waters Delta 600 chromatography system consisting of Waters UPLC H-class Core System (column Waters Acquity UPLC BEH C18 1.7 mm, 2.1 × 100 mm), Waters Acquity UPLC PDA detector, and mass spectrometer Waters SQD2. LC method A was used (eluent H 2 O modified with 0.1% formic acid/MeCN modified with 0.1% formic acid, gradient 0�100%, run-length 7 min) unless LC method B was used as noted (eluent H 2 O/MeCN, gradient 0�100%, run-length 7 min) and the MS method (ESI + and/or ESI − , cone voltage = 30 V, mass detector range 100−1000 Da) was used. Flash chromatography was performed on a Teledyne ISCO CombiFlash Rf + automated chromatography system using FLUKA silica gel 60 Å (230−400 mesh) for normal phase chromatography and RediSep RF Gold C18 Aq columns (20−40 μm, TELEDYNE ISCO) for reverse-phase chromatography. Preparative TLC (pTLC) was performed on Preparative UNIPLATES from ANALTECH (20 × 20 cm, 2 mm thickness, Catalog number 02015). The high-resolution mass spectra were measured on an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific) using ESI + , EI + , or CI + ionization. NMR spectra were measured on Bruker Advance III HD 400, 500, and 600 instruments: 1 H NMR spectra at 400.1, 499.9, and 600.1 MHz, respectively, and 13 C NMR spectra at 100.6 MHz, 125.7 MHz, and 150.9 MHz, respectively. 1 H NMR spectra are reported relative to the residual solvent (δ = 7.26 for CHCl 3 and δ = 2.50 for DMSO) and 13 C NMR spectra are referenced relative to the solvent signal (δ = 77.16 for CDCl 3 and δ = 39.70 for DMSO-d 6 ). APT, COSY, HSQC, and HMBC spectra were used to aid full assignment of NMR signals.
The numbering system used for the description of the NMR spectra of the prepared compounds is depicted in Figure 3.
Data for 1 H NMR spectra are reported as follows: chemical shift in ppm (multiplicity, coupling constant in Hz, integration, assignment) and for 13 C NMR spectra as follows: chemical shift in ppm (assignment). Multiplicity and qualifier abbreviations are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and b = broad. All compounds are >95% pure by HPLC analysis.

Synthetic Procedures. General Procedure for Introduction of Arm II (Method A).
Corresponding chloropyrimidine (1 equiv) and 4aminobenzonitrile (1.1 equiv) were suspended in i-PrOH (0.08 M, degassed by bubbling with Ar for 15 min) in a microwave vial. The vial was sealed and heated to 150°C for 0.5−1.5 h in the microwave reactor. The volatiles were removed, and the residue was purified by flash chromatography.
General Procedure for Introduction of Arm II followed by Reductive Cyclization (Method B). Corresponding chloropyrimidine (1 equiv) and 4-aminobenzonitrile (1.1 equiv) were suspended in i-PrOH (0.1 M, degassed by bubbling with Ar for 15 min) in a microwave vial. The vial was sealed and heated to 150°C for 2 h in the microwave reactor. The volatiles were removed, and the residue was purified by flash chromatography (50 g C18-SiO 2 , 0−100% MeOH/H 2 O, min. 5 column volumes (CV) 100% MeOH) fractions containing the desired nitriles were combined, evaporated, and used directly in the next step. The obtained nitriles, SnCl 2 (5 equiv) and Sc(OTf) 3 (0.2 equiv), were dissolved in EtOH (0.01 M) and H 2 O (10 equiv) was added. The reaction mixture was heated to 50°C for 18−20 h. The volatiles were evaporated and the residue purified by flash chromatography.
General Procedure for Reduction of the Nitro Group with SnCl 2 (Method C). The corresponding nitropyrimidine (1 equiv) and SnCl 2 (10 equiv) were dissolved in EtOH (0.05 M) and H 2 O (20 equiv), and the mixture was heated to 60°C for 18 h. The volatiles were evaporated, and the residue was purified by flash chromatography (50 g C18-SiO 2 , 0−100% MeOH/H 2 O, min. 5 CV 100% MeOH) to give the amine.
General Procedure for Cyclization with CDI (Method D). To the corresponding amine (1 equiv) dissolved in DCM (0.075 M) was portionwise added CDI (2.2 equiv) at 25°C. The mixture was stirred at 25°C for 2 h. Work-up is given for individual compounds.
General Procedure for Removal of the TIPS Protecting Group (Method E). To the corresponding silyl ether (1 equiv) dissolved in THF (0.04 M) cooled to 0°C (ice/water bath) was added TBAF (1 M in THF, 1.2 equiv), and the cooling bath was removed. The mixture was stirred at 25°C for 4 h. The volatiles were evaporated, and the residue was purified by flash chromatography (50 g C18-SiO 2 , 0−100% MeOH/H 2 O) to give the corresponding alcohol.
General Procedure for Parikh−Doering Oxidation (Method F). The corresponding alcohol (1 equiv), DMSO (20 equiv), and DIPEA (5 equiv) were dissolved in THF (0.025 M), and the mixture was cooled to −10°C (NaCl/ice/water bath). Sulfur trioxide pyridine complex (14 equiv) was added in a spatulatipwise manner over 10 min, and the mixture was stirred at −10°C for an additional 20 min, after which time UPLC−MS indicated full consumption of the starting material. The reaction was quenched by H 2 O (1 mL), allowed to warm to 25°C, and the volatiles were evaporated. The residue was purified by flash chromatography (30 g C18-SiO 2 , 0−100% MeCN/ H 2 O, loaded in 1 mL DMF) to give the corresponding aldehyde.

Ethyl N-(4-Cyano-2,6-dimethylphenyl)-N-(2-((4-cyanophenyl)amino)-5-nitropyrimidin-4-yl)glycinate
MDCK Assay. cMdr1-KO cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with sodium pyruvate and GlutaMax, supplemented with 1% Pen/Strep and 10% fetal bovine serum in an incubator set at 37°C, 90% humidity, and 5% CO 2 . MDCKII-cMdr1-KO cells were grown to confluence over 3 days on 96-well PET plates with 1 μm pore size, polyester membrane (Corning 3392). Experiments were run using HBSS donor buffer from Invitrogen containing an additional 10 mM HEPES, 15 mM glucose, and 0.1% BSA adjusted to pH 7.4. The receiver well had HBSS buffer supplemented with 1% BSA, 10 mM HEPES, 15 mM glucose, and the pH was adjusted to 7.4. TEER values were read to test membrane integrity at the beginning of the assay. The experiment was started by the addition of dosing solutions containing test compounds. Samples were taken from the donor compartment at 0/ 120 min and from the receiver compartment at 120 min. Each compound was tested in two separate replicate wells. All samples were immediately collected in a 72:8:20 MeCN/MeOH:H 2 O mix to precipitate protein and stabilize the test compounds. Cells were dosed on the apical side to determine forward (A to B) permeability. To test for non-specific binding and compound instability, the total amount of drug was quantified at the end of the experiment and compared to the material present in the original dosing solution as a percentage of recovery. Samples were analyzed by RF-QToF.
Measurement of Predicted Clearance Values in HLM. Metabolic stability of compounds was assessed using HLM (Corning cat. 457117). 52 In this assay, 10 nL compounds at a concentration of 1 mM in 100% DMSO are dispensed into 384-well polypropylene plates using the Echo 550 acoustic liquid dispenser (Labcyte). Each plate contains 384 wells with a single test compound in each well.
A solution of HLM at 2 mg/mL in 100 mM K 2 HPO 4 /KH 2 PO 4 pH 7.4 with 0.0225 mg/mL Alamethicin from Trichoderma viride (Sigma-Aldrich cat. A4665-10MG) was incubated on ice for 15 min. 5 μL of this solution was added to individual wells following 15 min incubation at room temperature; and supplemented with 5 μL NADPH Regenerating Solution of cofactors (Corning Gentest UGT Reaction Mix solutions A and B, cat. 451200 and 451220) containing 100 mM K 2 HPO 4 /KH 2 PO 4 pH 7.4, 2.6 mM NADP + , 6.6 mM glucose-6-phosphate, 6.6 mM MgCl 2 , 0.8 U/mL glucose-6-phosphate dehydrogenase, 0.1 mM sodium citrate, and 6.8 mM uridine diphosphate-glucuronic acid. The final concentration of analyte compounds at the beginning of the reactions was 1 μM. The reactions were incubated at 37°C, and time points of 0, 5, 15, 30, 40, 50, 60, and 70 min were collected for further analysis. Background data were collected using reactions without analyte compounds.
Upon collection of the reaction time points, samples were quenched at the set time points with 30 μL of a solution of 72% acetonitrile, 8% methanol, 0.1% formic acid, 19.9% water, and labetalol as an internal standard (IS) at a concentration of 650 nM. Reaction plates were span in a centrifuge at a speed of 4000 rcf for 30 min and 4°C, following a dilution of the 10 μL quenched reaction into 40 μL de-ionized water, yielding assay plates.
Samples were analyzed by a solid-phase extraction coupled with a quadrupole time-of-flight mass spectrometer, using an Agilent QToF 6530 RapidFire 360 system with C4 type A solid state cartridges. Analysis was performed in either positive or negative ionization modes. Mobile phases contained 0.1% formic acid in water for loading analytes onto solid state extraction cartridges, and 0.1% formic acid in acetonitrile for elution into the mass spectrometer in positive ionization mode, or 0.1% acetic acid in water for loading and 0.1% acetic acid in acetonitrile for extraction in negative ionization mode. Peak-area ratios of integrated counts for individual compounds to IS were plotted as a semi-logarithmic chart of log versus time. Initial, linear portion of decay was fitted to a linear regression equation to derive the half-time of a compound decay.
Pharmacological parameters for an analyte compound metabolism were calculated using the equations described in Table S2.
Solubility Assessment. Compound solubility was measured at Analiza (Cleveland, OH) by the standard shake-flask method at pH 1.2 and 7.4 and quantitated by HPLC-UV.
Protein Crystallization. HIV-1 Reverse Transcriptase (RT) was expressed and purified as described previously. 15 For crystallization trials, RT was concentrated to 20 mg/mL in a final buffer containing 10 mM Tris pH 7.0, 25 mM KCl, and 1 mM DTT. The compounds were dissolved into DMSO to make a stock solution at 10 mM and then mixed with RT for a final concentration of 400 μM. Crystals were obtained in 0.8−0.96 M K/Na tartrate and MES buffer pH 6.0 by hanging drop vapor diffusion at 20°C. Crystals were introduced to a cryoprotectant solution containing 20% glycerol in addition to the above concentrations of the mother liquor components. The crystals were then cooled in a bath of liquid nitrogen.
Data Collection, Model Building, and Refinement. Data was collected at The Advanced Light Source on BL5.0.2 at a temperature of 100 K. Diffraction data was processed with XDS. 53 The structure was determined by the molecular replacement method using the program Phenix 54 with PDB code 3MEE as a search model. Additionally, simulated annealing, energy minimization, and B-factor refinement were carried out in Phenix. 54 Model building was performed with the molecular graphics program Coot. 55 ■ ASSOCIATED CONTENT

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01574. 1 H NMR and 13 C NMR spectra of prepared compounds, HPLC chromatograms of in vitro evaluated compounds, data collection and refinement statistics for X-ray structures, and pharmacological parameters for an analyte compound metabolism (PDF) Molecular formula strings (CSV) Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Accession Codes
The atomic coordinates and structure factors have been deposited into the Protein Data Bank. The accession numbers are 8FCC, 8FCD, and 8FCE for compounds 2, 4, and 6, respectively. Authors will release the atomic coordinates upon article publication.